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1 Charité Universitätsmedizin Berlin, 12249 Berlin, Germany
2 Institut Pasteur, 75724 Paris, France
3 Baxter Vaccine AG, A-2304 Orth/Donau, Austria
Correspondence
Dania Richter
drichter{at}charite.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, with strain PC-Eq17N5T (=DSM 16813T=CIP 108855T) as the type strain.
Published online ahead of print on 9 December 2005 as DOI 10.1099/ijs.0.64050-0.
The GenBank/EMBL/DDBJ accession numbers for the fla, groEL, hbb, ospA, recA, rrs and rrf–rrl intergenic spacer gene sequences of Borrelia spielmanii sp. nov. PC-Eq17N5T are DQ133508, DQ133511, DQ133514, DQ133517, DQ133520, DQ133523 and DQ133526, respectively. Accession numbers for other gene sequences obtained in this study are given in Supplementary Table S2 in IJSEM Online.
Tables detailing similarity values for concatenated sequences of species within Borrelia burgdorferi s.l. and GenBank accession numbers for gene sequences derived in this study are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Borrelia garinii (Baranton et al., 1992) and Borrelia afzelii (Canica et al., 1993) cause Lyme disease in humans. Particular species, such as Borrelia japonica (Kawabata et al., 1993), ‘Borrelia andersonii’ (Marconi et al., 1995), ‘Borrelia bissettii’ (Postic et al., 1998), Borrelia sinica (Masuzawa et al., 2001), Borrelia turdi and Borrelia tanukii (Fukunaga et al., 1996), appear not to be pathogenic and have a restricted geographical distribution. Borrelia valaisiana (Wang et al., 1997), which is widely distributed in Europe and Asia, and Borrelia lusitaniae (Le Fleche et al., 1997), which infects vector ticks in Europe and North Africa (Younsi et al., 2005), are potentially pathogenic. DNA specific for B. valaisiana has been detected in human samples (Rijpkema et al., 1997; Diza et al., 2004) and a strain of B. lusitaniae has been isolated from the skin of a patient (Collares-Pereira et al., 2004). The most recent species described within the complex of B. burgdorferi s.l. was previously effectively published as ‘Borrelia spielmani’ Richter et al. 2004. Its exceptionally narrow specificity for a particular reservoir host, garden and hazel dormice, distinguishes it from all other Lyme disease spirochaetes (Richter et al., 2004). This unique biological relationship, together with its genotypic and phenotypic characteristics (Van Dam et al., 1993; Wang et al., 1999; Derdáková et al., 2003; Richter et al., 2004), suggested that this dormouse-associated spirochaete constitutes a distinct novel species. However, because of the lack of DNA–DNA hybridization data, the name of this species has not been validly published. Current practice requires that validation of a novel bacterial species should be based on the level of DNA–DNA reassociation by means of whole DNA–DNA hybridization (WDDH), which is considered to be the gold standard in taxonomy (Wayne et al., 1987). Few laboratories, however, can perform this technique, because it is non-cumulative and must include reference strains for all known species. Moreover, this method is not applicable for bacteria that cannot be cultivated and its application for slow-growing bacteria is limited. In addition, WDDH data may not be reproducible (Stackebrandt et al., 2002). Thus, alternative methods that can replace DNA–DNA reassociation are required (Stackebrandt et al., 2002).
The rapid development of sequencing techniques has made their application inexpensive and permits structural analysis of bacterial populations (Achtman, 2004). Multilocus sequence typing (MLST) is based on the identification of allelic mismatches of usually seven housekeeping genes (Selander et al., 1986; Gevers et al., 2005). It has been used to reveal the population structure of diverse prokaryotic organisms at the intraspecific level, including B. burgdorferi s.s. and B. afzelii (Bunikis et al., 2004; Qiu et al., 2004). Recently, it was proposed that multilocus sequence analysis (MLSA), a phylogenetic characterization using sequences of alleles of several genes, should be applied for species delineation (Gevers et al., 2005). For MLSA, sequences are processed using a distance method procedure rather than the cluster analysis procedure used in MLST. This tool may replace DNA–DNA reassociation, provided that both techniques demonstrate a sufficient degree of congruence (Stackebrandt et al., 2002). We have previously accumulated values for DNA–DNA reassociation of all type strains and various other strains of B. burgdorferi s.l. by using the S1-TCA method (Grimont et al., 1980).
To validate the MLSA method for species delineation, we sequenced seven loci of numerous B. burgdorferi s.l. strains, including type strains, and compared the results with our extensive data collection of DNA–DNA similarity and thermal stability. MLSA was subsequently applied to several isolates of the recently described, but not yet validly published, species ‘B. spielmani’ Richter et al. 2004.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) belong to five species endemic to Europe, B. burgdorferi s.s., B. garinii, B. afzelii, B. valaisiana and B. lusitaniae. Information regarding DNA–DNA reassociation was available for each of these strains (Postic et al., 1990; Baranton et al., 1992; Canica et al., 1993; Le Fleche et al., 1997; Wang et al., 1997) and sequences at several of the selected loci were accessible from the literature or databases (Postic et al., 1994; Le Fleche et al., 1997; Valsangiacomo et al., 1997; Wang et al., 1997; Casati et al., 2004; Park et al., 2004). For the delineation of ‘B. spielmani’ by MLSA, we used the first isolate described of this group, strain A14S, which was isolated from the erythema migrans of a patient, and three strains, including the type strain PC-Eq17N5T, derived from Ixodes ricinus ticks that had fed on each of three garden dormice (Eliomys quercinus), which had been live-trapped in Alsace, France (Richter et al., 2004), as well as two isolates obtained from acute skin lesions of a Danish and a Hungarian patient. DNA of each of these strains was extracted from the centrifugation pellet of cultivated isolates, either by boiling at 100 °C for 10 min or by using a QIAamp DNA mini kit (Qiagen).
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). All loci were amplified by a single PCR. The reactions were performed in a final volume of 50 µl, comprising 0·2 µM of each primer of a primer pair, 200 µM of each dNTP, 1·25 or 1 U Taq polymerase (QBiogene or Qiagen, respectively) and 1x Taq buffer (1·5 mM MgCl2). The mixture was placed in a thermocycler, heated to 93 °C for 1 min and subjected to 35 cycles of denaturation for 1 min at 93 °C, annealing for 1 min at either 51 °C for rrs and groEL loci or 59 °C for the remaining loci and extension for 1 min at 72 °C, followed by a final extension step at 72 °C for 5 min. The products were sequenced either by Genome Express (Meylan, France) or by using the dideoxynucleotide chain-termination method on a Licor DNA4200 sequencer with the same primers as for PCR.
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) algorithm was used for sequence alignments and MEGA 3 (Kumar et al., 1993) software for phylogenetic analyses of both individual and concatenated sequences. Distances were calculated by using the Jukes and Cantor correction (Saitou & Nei, 1987) in a pairwise deletion procedure. A similarity table was generated from a distance matrix (P distance values) with Excel. Unweighted pair group with mathematical average (UPGMA) (Sneath & Sokal, 1962) and neighbour-joining (NJ) (Saitou & Nei, 1987) methods were both used to construct phylogenetic trees. Percentage support values were obtained using a bootstrap procedure. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or NJ (data not shown), sequences from isolates belonging to each species with a validly published name constituted a single cluster on each individual tree. The rrf–rrl intergenic spacer provided greater strain diversity within a species than did other loci. B. lusitaniae strains PotiB1 and PotiB2T were particularly divergent at this locus, although their rrf–rrl sequences were clearly distinct from those of other species. Otherwise, B. garinii clearly appears to be the most diverse species, because the strain clustering varied according to the locus targeted.
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), supported by bootstrap analysis. Similar results were obtained by using both UPGMA (Fig. 2a) and NJ (Fig. 2b) methods.
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, 1994; Baranton et al., 1992). The clustering of strains obtained by MLSA strongly associated with the clustering inferred from DNA–DNA relatedness values (Fig. 3). Within a species, defined by values of DNA–DNA relatedness above 70 % and a thermal stability estimated at 
Tm below 5 °C (Wayne et al., 1987), sequence similarity values deduced from genetic distances ranged from 97·9 to 99·8 % (Supplementary Table S1 in IJSEM Online). In contrast, sequence similarity values compared across species ranged from 92 to 94·9 % (Supplementary Table S1 in IJSEM Online). A slight interspecific discrepancy was observed when strains B. afzelii VS461T and B. garinii PBi were compared. Although the similarity value was 94 %, the DNA–DNA relatedness reached 74 %. However, a Tm value of 9 °C demonstrated that these strains comprised two distinct species, as did the MLSA similarity value determined in this study. An MLSA similarity value of 97·9 %, therefore, appears to be useful as a cut-off to differentiate B. burgdorferi s.l. species.
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). As for all other delineated species, the rrf–rrl locus varied most. Sequences were scattered among three branches when published rrf–rrl sequences of ‘B. spielmani’ isolates derived from either ticks [GenBank accession nos AY147009 (PC-Eq17N5T), AY573193 and AF497994] or human samples (accession no. U76616) and unpublished sequences of isolates obtained in our laboratories from I. ricinus (PC-Eq2/1, PC-Eq2r and 4AJL150) or human skin (2102, PZ30802, DK35 and DK38) were included in the analysis. In contrast, the sequences of ‘B. spielmani’ were identical for the conserved fla locus and, more unexpectedly, were virtually identical for the ospA locus. A very similar clustering resulted from the phylogenetic analysis of the groEL, hbb and recA loci.
When concatenated sequences were analysed, isolates of ‘B. spielmani’ appeared to be closely related to one another, but unambiguously diverged from all other species that were included in this study (Fig. 2). When we applied the MLSA similarity cut-off of 97·9 % to ‘B. spielmani’, all examined isolates constituted a single and homogeneous species, with similarities ranging from 99·2 to 100 %, while differing from other species by similarity values of 92·1–94·8 % (Supplementary Table S1 in IJSEM Online).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover, several methodologies showed a good correlation with WDDH data. For example, analysis of B. burgdorferi s.l. strains by multilocus enzyme electrophoresis (MLEE; Boerlin et al., 1992) produced values that strongly correlated to those of DNA–DNA reassociation (Baranton et al., 1992). Even arbitrarily primed PCR (Welsh et al., 1992) suggested that isolate DN127 differed from other previously studied isolates and may be a member of a novel species. Indeed, subsequently, isolate DN127 was proposed as the type strain of ‘B. bissettii’ (Postic et al., 1998). However, methods involving multiple loci distributed on the whole genome of an organism provide a more valuable alternative to laborious DNA–DNA reassociation (Stackebrandt et al., 2002). MLST and recently, more appropriately, MLSA, have been proposed as true alternatives to DNA–DNA reassociation for taxonomic purposes (Lan & Reeves, 2001; Stackebrandt et al., 2002; Gevers et al., 2005). Well-defined conditions for MLST have to be met, in particular the requirement for a minimum of five housekeeping genes under stabilizing selection, in order to obtain an informative level of data (Stackebrandt et al., 2002). Whereas MLST characterizes genotypic relationships of prokaryotes at an intraspecific level by identifying allelic mismatches of housekeeping genes, MLSA permits phylogenetic characterization by yielding clusters of concatenated sequences of multiple genes (Gevers et al., 2005). In our MLSA study, we used seven loci whose relevance for taxonomic studies of B. burgdorferi s.l. had been demonstrated previously. Five of them are located on the chromosome and therefore evolved in a clonal way, while not being subjected to lateral transfer (Dykhuizen et al., 1993): rrs gene (Le Fleche et al., 1997; Wang et al., 1997; Postic et al., 1998), fla gene (Fukunaga & Koreki, 1996), groEL gene (Park et al., 2004), hbb gene (Valsangiacomo et al., 1997) and recA gene (Casati et al., 2004). Instead of operational genes, we chose informational genes (rrs, hbb, groEL, recA) that are embedded in a network of interactions and are subjected only minimally to lateral transfer (Jain et al., 1999). We added the fla gene, encoding flagellin, because it constitutes the endoflagella of spirochaetes and therefore is not targeted by selective pressure of the host's immune response. The ospA locus, on the other hand, represents an adaptive, plasmid-encoded gene, which has been employed previously for taxonomic purposes (Dykhuizen et al., 1993; Bunikis et al., 2004). Although this gene may be subject to lateral transfer, it occurs only rarely (Dykhuizen et al., 1993). In fact, the tree generated from the ospA gene appeared to evolve clonally for all sequences studied. Finally, we chose the non-coding locus rrf–rrl intergenic spacer, as its sequence correlates well taxonomically (Postic et al., 1994). These last two loci represent the fast-evolving part of the genome, contrasting with the other loci. Independent of the locus considered, the phylogeny reflects taxonomic associations, thereby confirming that B. burgdorferi s.l. evolves clonally (Dykhuizen et al., 1993).
MLST was originally designed for epidemiological purposes (Achtman, 2004). The related, but phylogenetic, approach, which was recently termed MLSA (Gevers et al., 2005), has been used successfully to delineate species of highly recombinogenic bacteria (Hanage et al., 2005). We demonstrate here for B. burgdorferi s.l. that sequence similarities derived by MLSA strictly correlate with data inferred from WDDH. Relatedness deduced from MLSA, therefore, is applicable for distinguishing B. burgdorferi s.l. species, if performed with a sufficient number of appropriately selected loci. Species delineation by MLSA must be based on a cut-off in sequence similarity that is determined by comparing genetic distances obtained from both DNA–DNA reassociation and MLSA. As the similarity cut-off, we selected the widest genetic distance corresponding to the lowest percentage similarity recorded within any of the previously recognized B. burgdorferi s.l. species. Phylogenetic analyses of concatenated sequences in a bootstrap procedure provided additional support of the robustness of MLSA. Therefore, MLSA constitutes a valuable alternative to laborious DNA–DNA hybridization.
Using MLSA, we confirmed the status of a previously delineated species among the B. burgdorferi s.l. complex, ‘B. spielmani’. This species is characterized by a peculiar reservoir relationship. In nature, strains of ‘B. spielmani’ perpetuate in garden and hazel dormice (Richter et al., 2004). No other member of the B. burgdorferi s.l. complex is restricted to such a narrow host range. This unique ecological niche together with its genotypic and phenotypic characteristics (Van Dam et al., 1993; Wang et al., 1998; Derdáková et al., 2003; Richter et al., 2004) distinguish it from all other spirochaetes causing Lyme disease. The distinctiveness of ‘B. spielmani’ is fully reflected in our MLSA analysis.
Numerous questing I. ricinus ticks and those feeding on dormice have been found to harbour strains of ‘B. spielmani’ in France (Richter et al., 2004). Questing ticks infected by this dormouse-associated spirochaete have also been collected in the Czech Republic (Derdáková et al., 2003), Russia (GenBank accession no. AY573193) and Germany (Rauter et al., 2002). ‘B. spielmani’ causes erythema migrans in humans and was isolated from or detected in patients' skin in the Netherlands (Van Dam et al., 1993), Slovenia (E. Ruzic-Sabljic, unpublished data), Hungary (Földvári et al., 2005), twice in Denmark (I. Livey, unpublished data) and twice in Germany (Michel et al., 2004). Hitherto, ‘B. spielmani’ has been detected in ticks and patients solely in Europe; its distribution may correspond closely to that of dormice.
Description of Borrelia spielmanii sp. nov.
Borrelia spielmanii (spi.el.man'i.i. N.L. gen. n. spielmanii of Spielman, named in honour of Andrew Spielman, who described for the first time the life cycle and biological relationships of B. burgdorferi s.l.).
Previously effectively published as ‘Borrelia spielmani’ Richter et al. 2004. The description is given in Richter et al. (2004). Note that the spelling of the name B. spielmanii has been corrected. The type strain is PC-Eq17N5T (=DSM 16813T=CIP 108855T).
Taken together, the MLSA results agree fully with data obtained by WDDH. MLSA therefore constitutes a valuable alternative for a reliable and precise delineation of B. burgdorferi s.l. species. This methodology results in solid sequence data with a highly discriminatory power that are not subject to experimental variation and may easily be shared, allowing interlaboratory comparison. It is important that this technique is widely adopted, because it facilitates studies on various bacterial isolates that hitherto remain unclassified due to the impediment of cumbersome methods.
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ACKNOWLEDGEMENTS |
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) and between (
) species. Values smaller than a genetic distance of 0·3 determined by WDDH generally designate an intraspecific relationship (Wayne et al., 1987
) constitutes an exception.